Intrabodies

Question 1

What is an intrabody?

Answer 1

An antibody that is expressed within the intracellular environment. Functions to modify or interrupt biological processes of intracellular proteins.

Question 2

What kind of antibodies are intrabodies?

Answer 2

scFvs and sD Ab

Question 3

How do we produce intrabodies?

Answer 3

Clone V region genes into viral vector (linearised baculovirus, adenovirus, retrovirus) and then insect cells

Question 4

How can we target intrabodies to specific locations in the cell?

Answer 4

Intracellular targeting motifs - NLS/KDEL

Question 5

What are the functions of intrabodies?

Answer 5

Sequester
Modulate enzymes
Apoptosis 
Degradation
Cellular signalling

Question 6

Discuss an example of how intabodies can degrade proteins?

Answer 6

Anti-gephyrin scFv joined to E3 ligase - targets gephyrin for degradation by the proteasome. Used to ablate inhibitory synapses

Question 7

How can intrabodies be used in cancer therapy?

Answer 7

Target p53/Ras/Rb for degradation

Sequester erb-2 in breast cancer - gene over expressed in 30% of breast cancers - encodes a GP involved in the EGFR

Question 8

How can intrabodies be used in HIV therapy?

Answer 8

Target CCR5 (entry in cell) and gp120 (HIV envelope)

Question 9

What modifications can we make to intrabodies to increase their effectiveness?

Answer 9

Attach to localisation sequence
Modification of VL for hyper stability
Selection of Ab resistant to intracellular environment
Expression as fusion protein with MBP to stabilise intracellularly

Question 10

Discuss how intrabodies can be used against parkinson’s?

Answer 10

Against alpha-synuclein (α-syn) - misfolding causes familial PD. While results are promising, scFvs against the target have low intracellular affinity